Characterization and partial purification of soluble, lysosomal phospholipase(s) A2 from adrenal medulla.
Soluble, cation-dependent, lysosomal phospholipase A2 in bovine adrenal medulla has been biochemically characterized and partially purified, and its unique pH-dependent modulation by cations has been investigated. Chromatographically distinct activities with somewhat broad pI ranges centered at 7.8, 8.1, and 8.4 have been purified 83-, 1900- and 4400-fold, respectively, from the soluble fraction of tissue homogenates. With a specific activity of 4.2 mumol phospholipid hydrolyzed per mg protein per min, the fraction of pI 8.4 is the most highly purified lysosomal phospholipase A2 reported to date; yet silver staining of isoelectric focusing gels indicates that all three species are still only minor components of the protein mixtures with which they co-purify. Lysosomal phospholipase(s) A2 has an apparent molecular weight of 30,600, as determined by gel permeation chromatography; and is probably an oligomannose-containing glycoprotein as indicated by binding to concanavalin A-Sepharose and elution by methyl alpha-D-mannopyranoside. Cation concentrations modulate hydrolysis of biomembranous phospholipid, but not neat liposomal phospholipids, in a complex manner over a broad pH range (pH 4.0-8.0). Triton X-100 stabilizes the enzyme(s) but is inhibitory when present during assay; consequently, detergent-phospholipid mixed micelles are poor substrates. Thus, experimental results are dramatically dependent on the physicochemical nature of the substrate. The role of this phospholipase(s) A2 in the membrane fusion and lysis events of catecholamine secretion, as well as its regulation by cellular proteins, can now be investigated utilizing this partially purified enzyme(s).
Bartolf
M
M
Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0164.
Franson
R C
RC
D000313:Adrenal Medulla / Q000201:enzymology*; D000818:Animals; D002412:Cations; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008247:Lysosomes / Q000201:enzymology; D010740:Phospholipases / Q000302:isolation & purification*; D010741:Phospholipases A / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D054467:Phospholipases A2
D002412:Cations; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2